Cellular Immunoregulatory Mechanisms in the Central Nervous System: Characterization of Noninflammatory and Inflammatory Cerebrospinal Fluid Lymphocytes Makoto Matsui, MD," Kazuhiro J. Mori, PhD,? and Takahiko Saida, MDS ~~~
Dual-label flow cytometric analysis of cerebrospinal fluid (CSF) and blood lymphocytes with combinations of monoclonal antibodies such as CD4 plus CD45R or Leu8, and CD8 plus CD11b was performed in 37 patients with noninflammatory neurological diseases (NINDs) to clarify the differences in cellular immunoregulatory mechanisms present in the central nervous system (CNS) and in the systemic circulation. In the CSF of patients with NINDs, the paucity of CD4+CD45R' and CD8+CD1l b + cells was striking, whereas the same subsets accounted for substantial proportions in the blood. CD4+CD45R- and CD4+Leu8- cells as well as CD8+CDllb- cells increased in the CSF when compared with those in the blood. Seven patients with active multiple sclerosis (MS) and 10 patients with other inflammatory diseases in the CNS (CNS-infl)were also studied. Patients with active MS were characterized by a consistent increase in percentage of CD4+CD45R- cells in the CSF, whereas an increase of CD4-CD45R+ cells in the CSF was a feature of the patients with CNS-infl, when compared with patients with NINDs. These findings indicate that the CNS is routinely surveyed by particular subsets of lymphocytes different from those in the blood, and cellular immune reaction in the CNS varies according to the types of CNS inflammatory conditions. Matsui M, Mori KJ, Saida T. Cellular immunoregulatory mechanisms in the central nervous system: characterization of noninflammatory and inflammatory cerebrospinal fluid lymphocytes. Ann Neurol 1990; 27647-651
The central nervous system (CNS) has long been thought to be an immunologically privileged site because of its relative unresponsiveness to allografts and inoculated pathogens C 11. The blood-brain barrier (BBB) isolates the CNS from the systemic circulation, so that only a few lymphocytes and very low levels of immunoglobulins are known to exist in the cerebrospinal fluid (CSF) in a noninflammatory state. Accumulating evidence has, however, indicated that lymphocytes in the CSF have unique immunological characteristics in comparison with those in the peripheral blood. For example, CSF lymphocytes proliferate poorly in response to plant lectins in inflammatory CNS diseases, whereas the blood lymphocytes examined simultaneously proliferate well C2, 31. It has been proposed that the CNS is routinely patrolled by activated T lymphocytes [4}. These findings indicate that a particular
population of T lymphocytes that are comparunentalized within the CNS play an important role in CNS immunoregulation. In the present study, we attempted to characterize T cell subsets in CSF from patients with both inflammatory and noninflammatory neurological disorders and compare the subsets with those in peripheral blood by using monoclonal antibodies (MoAbs) against CD45R C51 and Leu8 16, 71 antigens for CD4+ lymphocytes, and one MoAb against C D l l b 18, 91 antigen for CD8+ lymphocytes.
From the *Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto; the ?'Department of Biology, Faculty of Science, Niigata University, Niigata; and the %Department of Neurology, Utano National Hospital, Kyoto, Japan.
Received Jun 30, 1989, and in revised form Nov 14. Accepted for publication Dec 21, 1989. Address correspondence D~Matsui, do D~H, L,weiner, Center for Neurologic Diseases, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115.
Material and Methods Patients Thirty-seven patients with noninflammatory neurological diseases (NINDs) were studied to establish reference levels for the percentage of T-cell subsets in the CSF, as well as in the blood. This group consisted of 24 men and 13 women be-
Copyright 0 1990 by the American Neurological Association
647
tween 22 and 72 years of age (mean 45.4 yr), including 9 patients with spinocerebellar degeneration and other related disorders, 7 with motor neuron disease, 6 with noninflammatory neuropathy, 4 with remote cerebrovascular accidents, 4 with myopathy, 2 with myeloradiculopathy due to cervical spondylosis, and 5 with other illnesses. N o one in this group received corticosteroids or immunosuppressive agents. The number of CSF cells was less than 3/mm3 in every patient in the NINDs group. To clarify alterations in the CSF with inflammation, two groups of patients were examined. Seven patients with relapsing-remitting type of multiple sclerosis (MS group) were studied during the active stage of the disease, which was within 2 weeks of new neurological symptoms or worsening of previous ones, or within 2 weeks of peak deficit. They were patients with clinically definite or laboratory-supported definite MS, according to Poser's criteria [lo), ranging in age from 32 to 49 years (mean 39.9). Four showed a CSF pleocytosis (>5/mm3). Three of these 4 and 1 other patient in this group were being treated with corticosteroids. The other group consisted of 10 patients with inflammatory CNS diseases other than MS (CNS-infl), such as aseptic meningitis (3 patients), serous meningeal reaction to nonsuppurative pachymeningitis (2), meningoencephalitis (2), transverse myelitis (l),lupus meningitis (l), and syphilitic optic papillitis (1). The last 3 patients were receiving corticosteroids at the time of study. These patients were between 17 and 64 years of age (mean 34.0), and were associated with a slight to marked increase in number of CSF mononuclear cells (MNCs; 3-2000/mm3).
Cell Preparation, Staining and Flow Cytometry MNCs were prepared on the same day from the CSF obtained by atraumatic lumbar puncture and from the blood, as described previously [ll]. In brief, MNCs separated from the heparinized venous blood by Ficoll-Paque density-gradient centrifugation, or those collected from 8 to 16 ml of the CSF by a low-speed spinning at 4"C, were suspended in RPMI-1640 media supplemented with 10% fetal calf serum. To classify T-cell subsets, MoAbs such as fluorescein-conjugated CD3 (OKT3), CD4 (OKT4 or Leu3a), CD8 (OKT8 or Leu2a), CD19 (Leul2), anti-HLA-DR, CD45R (Leul8) [12], phycoerythrin (PE)-conjugated CD4 (Leu3), CD45R (2H4) [5], Leu8 [6], C D l l b (Leul5) [8}, and in a few instances, CDw29 (4B4) 1131 were studied. The OK series antibodies were purchased from Ortho Pharmaceuticals (Raritan, NJ); the Leu series and anti-HLA-DR antibodies were from Becton Dickinson (Mountain View, CA); 2H4 and 4B4 antibodies were from Coulter Immunology (Hialeah, FL). The MNCs were single-stained with CD3, CD8 (OKT8), CD19, or anti-HLA-DR MoAb, or doublestained with combinations of the previously mentioned antibodies such as CD4 plus CD45R or Leu8, and CD8 plus C D l l b , and less frequently CD4 plus CDw29. All stains were carried out at 4°C for 30 minutes. Flow cytometry of MNCs was performed by means of an Ortho Spectrum 111 (Ortho Diagnostic System, Westwood, MA). Monocytes and macrophages were gated out on the cytogram of the MNCs. At least 250 celldMoAb from the CSF and more than 5,000 cells/MoAb from the blood passed through a Spectrum 111. In a preliminary study on blood samples, the results were
648 Annals of Neurology Vol 27 N o 6 June 1990
Percentages of T-cell Subsets in Cerebrospinal Fluid and Peripheral Blood in Patients with NoninfEammatory Nelrrological Diseases"
CSF CD3+ CD4+CD45RCD4+CD45R+ CD4-CD45R' CD4 'Leu8CD4+Leu8+ CD4-Leu8' CD8+ CD8+CDllbCD8+CD1l b f CD8-CDllbt
PB
* 9.9;
88 55 f 1& 5 5 26 f 24 2 10 f 28 +24 t 1 +4 ?
69.4 f 7.5 26.7 7.0 17.1 & 5.0 43.3 7.1 8.6 f 3.3 33.1 +- 7.3 8.2 30.3 24.7 5 5.9 17.7 .+. 5.5 8.7 2 3.6 20.0 ? 5.2
* *
8.9 1.4' 5.5' 9.0b 9.8' 5.8' 7.0d 5.7d
*
l.lC
4.6'
"All values are expressed as mean
r
rt
0.381 0.236 0.261 0.181 0.061 -0.049 0.168 0.341 0.301 0.120 0.453
standard deviation. Exarnina-
tion by dual-labeling with CD8 and CDllb monoclonal antibodies were conducted in 10 of the 37 patients. bSignificantly increased (p < 0.001), as compared with PB. 'Significantly decreased (p < 0.001), as compared with PB. dSignificantly increased (p < 0.05), as compared with PB.
Statistical analysis of the data was performed by the Student's t test. Correlation coefficient ( r ) was evaluated by the least-square analysis. CSF
=
cerebrospinal fluid; PB
=
peripheral blood.
identical between OKT4 plus 2H4 and Leu3a plus Leu18 stains (data not shown). All results were expressed as percentage of each subset to total lymphocytes. Statistical analysis of the data was performed with the Student's t test. Correlation coefficient (r) of each lymphocyte subset between the CSF and the blood was computed by least-square analysis. Relationships between the T-cell subsets in the CSF or in the blood were also analyzed using the same method.
Results The proportions of T-cell subsets in a noninflammatory state are shown in the Table. The level for each subset in the CSF was significantly different from that in the blood. The percentage of CD19+ B cells was significantly lower in the CSF than in the blood (3% versus 10% as the mean percentage, p < O.OOl), whereas the proportions of HLA-DR antigen-positive (Ia+) lymphocytes were not different between the CSF and the blood (15% versus 16.9%). Lymphocytes bearing CD45R antigens were sparse in CSF, as compared with those in blood (6% versus 60.4%). Almost all the cells in the CD4+ population in the CSF lacked CD45R antigen, whereas half of the same population possessed Leu8 antigen. The CD 11b antigen-bearing cells in the CSF were significantly fewer than in the blood (5% versus 28.7%), so that most of the CD8+ cells in CSF were CD1 Ib-. A preliminary study indicated that the majority of the CD4+ lymphocytes in the CSF were CDw29+ (data not shown). No particular subset showed a significant correlation in percentages between CSF and blood (see Table). With respect
to the relationships between the subsets within the CD4 + population, a significant correlation was observed between the CD4+CD45R- and the CD4+ Leu8- cells in the CSF (Y = 0.699, p < 0.01). In the blood, the CD4+CD45R- population correlated with the CD4+Leu8- (r = 0.686, p < O.Ol), while the reciprocal CD4 +CD45Rf population correlated with the CD4+Leu8+ (r = 0.670, p < 0.01), and inversely with the CD4+Leu8- (Y = -0.671,p < 0.01). These results indicate that a substantial proportion of the CD4' cells in the blood expressed CD45R and Leu8 antigens simultaneously, whereas such cells were rare in the CSF. In contrast, the CD4+CD45RP population overlapped with the CD4+Leu8- in CSF as well as in blood. Since approximately 80% of the CD4+ cells in the blood were positive for Leu8, the CD4+CD4JRp Leu8 - population appeared to be larger in CSF than in blood. Examination of CSFs obtained from the patients with active MS revealed that the percentage of CD4 +CD45R- lymphocytes significantly increased (73 ? 4, p < O.OOl), while the patients with CNS-infl showed a significant increase in percentage of the CSF CD4-CD45R+ cells (18.9 & 8.9, p < 0.001) when compared with the patients with NINDs. Neither CSF pleocytosis nor corticosteroid treatment appeared to be linked to any particular alterations in CSF subsets (Figure). The CSF CD4+CD45R+ cells were consistently few; the mean percentages for these cells were 1.5% and 1.6% in the groups of patients with active MS and CNS-infl, respectively. The proportions of the other T-cell subsets in CSF were equivalent to those observed in the patients with NINDs (data not shown).
Discussion A prominent feature of the present study was the paucity of CD45R and C D l l b antigen-bearing cells in the CSF from neurological patients with noninflammatory states, in contrast to a relative preservation of the Leu8 antigen. The CD4+ lymphocytes, which simultaneously express high densities of CD45R antigens, have been reported to function as inducers of suppressor T cells 151, while the C D 8 + C D l l b + cells have been found to be suppressor cells 181. In this context, the CNS could be a hazardous site, susceptible to immune-mediated damage due to the lack of suppressor systems. However, a substantial number of Leu8 antigen-bearing CD4 + lymphocytes, proposed to be suppressor-inducer cells [6}, were observed in the CSF. A recent study showed that there is suppressor activity in CD8+ cells, irrespective of the presence or the absence of the CD11 molecule 1141. Therefore, it is possible that regulatory signals are given to the CD8+ suppressor-effector population by the CD4 +
I 1
+
a m t c l 0
30
"'k I I ACTIVE MS
CNS-infl
The percentages of CD4+CD45R- and CD4-CD45Rf cells in the cerebrospinalfluid (CSF). The cross-hatched areas represent the 2 S D range from the mean, evaluated in the 37 patients with noninflammatory neurological diseases. The triangles denote the presence of CSF pleocytosis ( > S l m d ) . The open and closed marks (circles or triangles) represent steroid-treatedand steroid-untreated patients, respectively. MS = multiple sclerosis; CNS-infl = patients with inflammatory diseases in the central nervous system other than MS.
Leu8+ suppressor-inducer subset in the CNS. Accumulating evidence has suggested that CD4 + lymphocytes lose highly expressed CD45R antigen when activated by stimulation with phytohemagglutinin 11517}, whereas the Leu8 antigen does not disappear permanently after prior activation {l8}. During this process, the CD4+ lymphocytes gain high densities of CDw29 1151 and UCHLl {l6} antigens. Since this phenotypic change was also observed in the CD8+ cell population { 151, the predominance of CD45R- cells in the CSF may indicate a sequestration of onceactivated lymphocytes into the CNS in a noninflammatory condition. This assumption agrees with the report that more than 80% of lymphocytes were both CDw29+ and UCHLl in CSF obtained from healthy volunteers 1191. A concordant finding is that numerous T a l + cells reside in the CSF of patients with NINDs 1203; the T a l antigen is a marker for previously activated T cells 121). It has been suggested that lymphocytes enter the CNS through postcapillary venules {I}, and only activated T cells can invade vas+
Matsui et al: Immunoregulation in the CNS 649
cular endothelium by degrading subendothelial extracellular matrix 122, 23). Thus, it is likely that activated T cells selectively cross the BBB and circulate through the CNS. An alternative interpretation, however, is that the CD45R-CDw29+UCHLl+ subset has a greater ability than CD45R+ cells to adhere to endothelial cells 1241. It is noteworthy that CD4+ CD45R-Leu8 lymphocytes are potent producers of interleukin-2 (IL-2) and interferon gamma upon activation 125, 261. Therefore, a relative enrichment of this type of cell in the CSF, although small in absolute number, seems to promote a swift immune response to target antigens. In the present study, the proportion of the CD4+ CD45R- cells in the CSF was significantly elevated in the active stage of relapsing-remitting MS, irrespective of ongoing steroid treatments. Although we classified MS disease activity according to time from neurological relapse without examining for new MS plaques by magnetic resonance imaging 1271, our criteria sufficed to select patients with active MS. The increased CD4+CD45R- cells appear to consist mainly of cells actively entering the CNS rather than those proliferating within the CNS, since IL-2 receptor-positive T cells have rarely been seen in the CSF or in the inflammatory lesions of MS 120, 28). By contrast, CD4CD45R+ cells were increased in the CSF of the patients with CNS-infl. A similar finding was that 2H4+ cells were numerous in tissues of viral encephalitis C291. Thus, there may be different patterns of cellular immune reaction in the CNS, according to the types of CNS inflammatory conditions. Further clarification of the specific functions of the subsets of lymphocytes compartmentalized within the CNS could serve as an important clue to immunoregulatory therapy for inflammatory CNS diseases.
This work was supported in part by a grant from the Ministry of Health and Welfare of Japan, and by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan (No. 01770542). We thank Mr S. Araya for technical assistance.
References 1. Leibowitz S, Hughes RAC. Immunity and the blood-brain barrier. In: Turk J, ed. Immunology of the nervous system. Current topics in immunology, vol 17. London: Arnold, 1983:l-19 2. Kam-Hanson S, Link H, Fryden A, Moller E. Reduced in vitro response of CSF lymphocytes to mitogen stimulation in multiple sclerosis. Scand J Immunol 1979;10:161-169 3. Hafler DA, Weiner HL. T cells in multiple sclerosis and inflammatory central nervous system diseases. Immunol Rev 1987; 100:307-332 4. Wekerle H , Lmington C, Lassmann H , Meyermann R. Cellular immune reactivity within the CNS. Trends Neurosci 1986;9: 271-277
650 Annals of Neurology Vol 27 No 6 June 1990
5. Morimoto C, Letvin NL, Distaso J, et al. The isolation and characterization of the human suppressor inducer T cell subset. J Immunol 1985;134;1508-15 15 6. Damle NK, Mohagheghpour N, Engleman EG. Soluble antigen-primed inducer T cells activate antigen-specific suppressor T cells in the absence of antigen-pulsed accessory cells: phenotypic definition of suppressor-inducer and suppressor-effector cells. J Immunol 1984;132:644-650 7. Kansas GS, Wood GS, Fishwild DM, Engleman EG. Functional characterization of human T lymphocyte subsets distinguished by monoclonal anti-Leu-8. J Immunol 1985;134:2995-3002 8. Landay A, Gartland GL, Clement LT. Characterization of a phenotypically distinct subpopulation of Leu-2 + cells that suppresses T cell proliferative responses. J Immunol 1983;131: 2757-2761 9. Clement LT, Dagg MK, Landay A. Characterization of human lymphocyte subpopulations: alloreactive cytoroxic T-lymphocyte precursor and effector cells are phenotypically distinct from Leu2+ suppressor cells. J Clin Immunol 1984;4:395-402 10. Poser CM, Paty DW, Scheinberg L, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983;13:227-23 1 11. Matsui M, Mori KJ, Saida T, et al. The imbalance in CSF T cell subsets in active multiple sclerosis. Acta Neurol Scand 1988; 77:202-209 12. Lanier LL. Non-lineage, LFA-1 family, and leukocyte common antigens. In: McMichael AJ, Beverley PCL, Cobbold S, et al, eds. Leukocyte typing 111. White cell differentiation antigens. Oxford: Oxford University Press, 1987:796-800 13. Morimoto C, Letvin NL, Boyd AW, et al. The isolation and characterization of the human helper inducer T cell subset. J Immunol 1985;134:3762-3769 14. Takeuchi T, DiMaggio M, Levine H, et al. C D l l molecule defines two types of suppressor cells within the T8+ population. Cell Immunol 1988;111:398-409 15. Serra HM, Krowka JF, Ledbetter JA, Pilarski LM. Loss of CD45R (Lp220) represents a post-thymic T cell differentiation event. J Immunol 1988;140:1435-1441 16. Akbar AN, Terry L, Timms A, et al. Loss of CD45R and gain of UCHLl reactivity is a feature of primed T cells. J Immunol 1988;140:2171-2178 17. Clement LT, Yamashita N, Martin AM. The functionally distinct subpopulations of CD4 + helperhnducer T lymphocytes defined by anti-CD45R antibodies derive sequentially from a differentiation pathway that is regulated by activation-dependent post-thymic differentiation. J Immunol 1988;141:14641470 18. Kanof ME, James SP. Leu-8 antigen expression is dininished during cell activation but does not correlate with effector function of activated T lymphocytes. J Immunol 1988;140:37013706 19. Hedlund G, Sandberg-Wollheim M, Sjogren HO. Increased proportion of CD4+CDw29+CD45R-UCHL-l+ lymphocytes in the cerebrospinal fluid of both multiple sclerosis patients and healthy individuals. Cell Immunol 1989;118:406-4 12 20. Hafler DA, Fox DA, Manning ME, et al. In vitro activated T lymphocytes in the peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. N Engl J Med 1985;312:14051411 21. Hafler DA, Fox DA, Benjamin D, Weiner HL. Antigen reactive memory T cells are defined by Tal. J Immunol 1986;137: 4 14-4 18 22. Naparstek Y, Cohen IR, Fuks Z , Vlodavsky I. Activated T lymphocytes produce a matrix-degrading heparan sulphate endoglycosidase. Nature 1984;310:241-244 23. Savion N, Vlodavsky I, Fuks 2. Interaction of T lymphocytes
and macrophages with cultured vascular endothelial cells: attachment, invasion, and subsequent degradation of the subendothelid extracellular matrix. J Cell Physiol 1984;118:169-178 24. Pittalis C, Kingsley G, Haskard D, Panayi G. The preferential accumulation of helper-inducer T lymphocytes in inflammatory lesions: evidence for regulation by selective endothelial and homotypic adhesion. Eur J Immunol 1988;18:1397-1404 25. Dohlsten M, Hedlund G, Sjogren H - 0 , Carlsson R. Two subsets of human CD4+ T helper cells differing in kinetics and capacities to produce interleukin 2 and interferon-? can be defined by the Leu-18 and UCHLl monoclonal antibodies. Eur J Immunol 1988;18:1173- 1178 26. Hedlund G, Dohlsten M, Sjogren HO, Carlsson R. Maximal
interferon-gamma production and early synthesis of interleukin2 by CD4+CDw29-CD45R-p80- humanT lymphocytes. Immunology 1989;66:49-53 27. Isaac C, Li DKB, Genton M, et al. Multiple sclerosis: a serial study using MRI in relapsing patients. Neurology 1988;38: 1511-1515 28. Hayashi T,Morimoto C, Burks JS, et al. Dual-label immunocytochemistry of the active multiple sclerosis lesion: major histocompatibility complex and activation antigens. Ann Neurol 1988;24:523-531 29. Sobel RA, Hafler DA, Castro EE, et al. The 2H4 (CD45R) antigen is selectively decreased in multiple sclerosis lesions. J Immunol 1988;140:22 10-2214
Matsui et al: Immunoregulation in the CNS
651
Dual-label flow cytometric analysis of cerebrospinal fluid (CSF) and blood lymphocytes with combinations of monoclonal antibodies such as CD4 plus CD45R or Leu8, and CD8 plus CD11b was performed in 37 patients with noninflammatory neurological diseases (NINDs) to clarify the differences in cellular immunoregulatory mechanisms present in the central nervous system (CNS) and in the systemic circulation. In the CSF of patients with NINDs, the paucity of CD4+CD45R' and CD8+CD1l b + cells was striking, whereas the same subsets accounted for substantial proportions in the blood. CD4+CD45R- and CD4+Leu8- cells as well as CD8+CDllb- cells increased in the CSF when compared with those in the blood. Seven patients with active multiple sclerosis (MS) and 10 patients with other inflammatory diseases in the CNS (CNS-infl)were also studied. Patients with active MS were characterized by a consistent increase in percentage of CD4+CD45R- cells in the CSF, whereas an increase of CD4-CD45R+ cells in the CSF was a feature of the patients with CNS-infl, when compared with patients with NINDs. These findings indicate that the CNS is routinely surveyed by particular subsets of lymphocytes different from those in the blood, and cellular immune reaction in the CNS varies according to the types of CNS inflammatory conditions. Matsui M, Mori KJ, Saida T. Cellular immunoregulatory mechanisms in the central nervous system: characterization of noninflammatory and inflammatory cerebrospinal fluid lymphocytes. Ann Neurol 1990; 27647-651
The central nervous system (CNS) has long been thought to be an immunologically privileged site because of its relative unresponsiveness to allografts and inoculated pathogens C 11. The blood-brain barrier (BBB) isolates the CNS from the systemic circulation, so that only a few lymphocytes and very low levels of immunoglobulins are known to exist in the cerebrospinal fluid (CSF) in a noninflammatory state. Accumulating evidence has, however, indicated that lymphocytes in the CSF have unique immunological characteristics in comparison with those in the peripheral blood. For example, CSF lymphocytes proliferate poorly in response to plant lectins in inflammatory CNS diseases, whereas the blood lymphocytes examined simultaneously proliferate well C2, 31. It has been proposed that the CNS is routinely patrolled by activated T lymphocytes [4}. These findings indicate that a particular
population of T lymphocytes that are comparunentalized within the CNS play an important role in CNS immunoregulation. In the present study, we attempted to characterize T cell subsets in CSF from patients with both inflammatory and noninflammatory neurological disorders and compare the subsets with those in peripheral blood by using monoclonal antibodies (MoAbs) against CD45R C51 and Leu8 16, 71 antigens for CD4+ lymphocytes, and one MoAb against C D l l b 18, 91 antigen for CD8+ lymphocytes.
From the *Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto; the ?'Department of Biology, Faculty of Science, Niigata University, Niigata; and the %Department of Neurology, Utano National Hospital, Kyoto, Japan.
Received Jun 30, 1989, and in revised form Nov 14. Accepted for publication Dec 21, 1989. Address correspondence D~Matsui, do D~H, L,weiner, Center for Neurologic Diseases, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115.
Material and Methods Patients Thirty-seven patients with noninflammatory neurological diseases (NINDs) were studied to establish reference levels for the percentage of T-cell subsets in the CSF, as well as in the blood. This group consisted of 24 men and 13 women be-
Copyright 0 1990 by the American Neurological Association
647
tween 22 and 72 years of age (mean 45.4 yr), including 9 patients with spinocerebellar degeneration and other related disorders, 7 with motor neuron disease, 6 with noninflammatory neuropathy, 4 with remote cerebrovascular accidents, 4 with myopathy, 2 with myeloradiculopathy due to cervical spondylosis, and 5 with other illnesses. N o one in this group received corticosteroids or immunosuppressive agents. The number of CSF cells was less than 3/mm3 in every patient in the NINDs group. To clarify alterations in the CSF with inflammation, two groups of patients were examined. Seven patients with relapsing-remitting type of multiple sclerosis (MS group) were studied during the active stage of the disease, which was within 2 weeks of new neurological symptoms or worsening of previous ones, or within 2 weeks of peak deficit. They were patients with clinically definite or laboratory-supported definite MS, according to Poser's criteria [lo), ranging in age from 32 to 49 years (mean 39.9). Four showed a CSF pleocytosis (>5/mm3). Three of these 4 and 1 other patient in this group were being treated with corticosteroids. The other group consisted of 10 patients with inflammatory CNS diseases other than MS (CNS-infl), such as aseptic meningitis (3 patients), serous meningeal reaction to nonsuppurative pachymeningitis (2), meningoencephalitis (2), transverse myelitis (l),lupus meningitis (l), and syphilitic optic papillitis (1). The last 3 patients were receiving corticosteroids at the time of study. These patients were between 17 and 64 years of age (mean 34.0), and were associated with a slight to marked increase in number of CSF mononuclear cells (MNCs; 3-2000/mm3).
Cell Preparation, Staining and Flow Cytometry MNCs were prepared on the same day from the CSF obtained by atraumatic lumbar puncture and from the blood, as described previously [ll]. In brief, MNCs separated from the heparinized venous blood by Ficoll-Paque density-gradient centrifugation, or those collected from 8 to 16 ml of the CSF by a low-speed spinning at 4"C, were suspended in RPMI-1640 media supplemented with 10% fetal calf serum. To classify T-cell subsets, MoAbs such as fluorescein-conjugated CD3 (OKT3), CD4 (OKT4 or Leu3a), CD8 (OKT8 or Leu2a), CD19 (Leul2), anti-HLA-DR, CD45R (Leul8) [12], phycoerythrin (PE)-conjugated CD4 (Leu3), CD45R (2H4) [5], Leu8 [6], C D l l b (Leul5) [8}, and in a few instances, CDw29 (4B4) 1131 were studied. The OK series antibodies were purchased from Ortho Pharmaceuticals (Raritan, NJ); the Leu series and anti-HLA-DR antibodies were from Becton Dickinson (Mountain View, CA); 2H4 and 4B4 antibodies were from Coulter Immunology (Hialeah, FL). The MNCs were single-stained with CD3, CD8 (OKT8), CD19, or anti-HLA-DR MoAb, or doublestained with combinations of the previously mentioned antibodies such as CD4 plus CD45R or Leu8, and CD8 plus C D l l b , and less frequently CD4 plus CDw29. All stains were carried out at 4°C for 30 minutes. Flow cytometry of MNCs was performed by means of an Ortho Spectrum 111 (Ortho Diagnostic System, Westwood, MA). Monocytes and macrophages were gated out on the cytogram of the MNCs. At least 250 celldMoAb from the CSF and more than 5,000 cells/MoAb from the blood passed through a Spectrum 111. In a preliminary study on blood samples, the results were
648 Annals of Neurology Vol 27 N o 6 June 1990
Percentages of T-cell Subsets in Cerebrospinal Fluid and Peripheral Blood in Patients with NoninfEammatory Nelrrological Diseases"
CSF CD3+ CD4+CD45RCD4+CD45R+ CD4-CD45R' CD4 'Leu8CD4+Leu8+ CD4-Leu8' CD8+ CD8+CDllbCD8+CD1l b f CD8-CDllbt
PB
* 9.9;
88 55 f 1& 5 5 26 f 24 2 10 f 28 +24 t 1 +4 ?
69.4 f 7.5 26.7 7.0 17.1 & 5.0 43.3 7.1 8.6 f 3.3 33.1 +- 7.3 8.2 30.3 24.7 5 5.9 17.7 .+. 5.5 8.7 2 3.6 20.0 ? 5.2
* *
8.9 1.4' 5.5' 9.0b 9.8' 5.8' 7.0d 5.7d
*
l.lC
4.6'
"All values are expressed as mean
r
rt
0.381 0.236 0.261 0.181 0.061 -0.049 0.168 0.341 0.301 0.120 0.453
standard deviation. Exarnina-
tion by dual-labeling with CD8 and CDllb monoclonal antibodies were conducted in 10 of the 37 patients. bSignificantly increased (p < 0.001), as compared with PB. 'Significantly decreased (p < 0.001), as compared with PB. dSignificantly increased (p < 0.05), as compared with PB.
Statistical analysis of the data was performed by the Student's t test. Correlation coefficient ( r ) was evaluated by the least-square analysis. CSF
=
cerebrospinal fluid; PB
=
peripheral blood.
identical between OKT4 plus 2H4 and Leu3a plus Leu18 stains (data not shown). All results were expressed as percentage of each subset to total lymphocytes. Statistical analysis of the data was performed with the Student's t test. Correlation coefficient (r) of each lymphocyte subset between the CSF and the blood was computed by least-square analysis. Relationships between the T-cell subsets in the CSF or in the blood were also analyzed using the same method.
Results The proportions of T-cell subsets in a noninflammatory state are shown in the Table. The level for each subset in the CSF was significantly different from that in the blood. The percentage of CD19+ B cells was significantly lower in the CSF than in the blood (3% versus 10% as the mean percentage, p < O.OOl), whereas the proportions of HLA-DR antigen-positive (Ia+) lymphocytes were not different between the CSF and the blood (15% versus 16.9%). Lymphocytes bearing CD45R antigens were sparse in CSF, as compared with those in blood (6% versus 60.4%). Almost all the cells in the CD4+ population in the CSF lacked CD45R antigen, whereas half of the same population possessed Leu8 antigen. The CD 11b antigen-bearing cells in the CSF were significantly fewer than in the blood (5% versus 28.7%), so that most of the CD8+ cells in CSF were CD1 Ib-. A preliminary study indicated that the majority of the CD4+ lymphocytes in the CSF were CDw29+ (data not shown). No particular subset showed a significant correlation in percentages between CSF and blood (see Table). With respect
to the relationships between the subsets within the CD4 + population, a significant correlation was observed between the CD4+CD45R- and the CD4+ Leu8- cells in the CSF (Y = 0.699, p < 0.01). In the blood, the CD4+CD45R- population correlated with the CD4+Leu8- (r = 0.686, p < O.Ol), while the reciprocal CD4 +CD45Rf population correlated with the CD4+Leu8+ (r = 0.670, p < 0.01), and inversely with the CD4+Leu8- (Y = -0.671,p < 0.01). These results indicate that a substantial proportion of the CD4' cells in the blood expressed CD45R and Leu8 antigens simultaneously, whereas such cells were rare in the CSF. In contrast, the CD4+CD45RP population overlapped with the CD4+Leu8- in CSF as well as in blood. Since approximately 80% of the CD4+ cells in the blood were positive for Leu8, the CD4+CD4JRp Leu8 - population appeared to be larger in CSF than in blood. Examination of CSFs obtained from the patients with active MS revealed that the percentage of CD4 +CD45R- lymphocytes significantly increased (73 ? 4, p < O.OOl), while the patients with CNS-infl showed a significant increase in percentage of the CSF CD4-CD45R+ cells (18.9 & 8.9, p < 0.001) when compared with the patients with NINDs. Neither CSF pleocytosis nor corticosteroid treatment appeared to be linked to any particular alterations in CSF subsets (Figure). The CSF CD4+CD45R+ cells were consistently few; the mean percentages for these cells were 1.5% and 1.6% in the groups of patients with active MS and CNS-infl, respectively. The proportions of the other T-cell subsets in CSF were equivalent to those observed in the patients with NINDs (data not shown).
Discussion A prominent feature of the present study was the paucity of CD45R and C D l l b antigen-bearing cells in the CSF from neurological patients with noninflammatory states, in contrast to a relative preservation of the Leu8 antigen. The CD4+ lymphocytes, which simultaneously express high densities of CD45R antigens, have been reported to function as inducers of suppressor T cells 151, while the C D 8 + C D l l b + cells have been found to be suppressor cells 181. In this context, the CNS could be a hazardous site, susceptible to immune-mediated damage due to the lack of suppressor systems. However, a substantial number of Leu8 antigen-bearing CD4 + lymphocytes, proposed to be suppressor-inducer cells [6}, were observed in the CSF. A recent study showed that there is suppressor activity in CD8+ cells, irrespective of the presence or the absence of the CD11 molecule 1141. Therefore, it is possible that regulatory signals are given to the CD8+ suppressor-effector population by the CD4 +
I 1
+
a m t c l 0
30
"'k I I ACTIVE MS
CNS-infl
The percentages of CD4+CD45R- and CD4-CD45Rf cells in the cerebrospinalfluid (CSF). The cross-hatched areas represent the 2 S D range from the mean, evaluated in the 37 patients with noninflammatory neurological diseases. The triangles denote the presence of CSF pleocytosis ( > S l m d ) . The open and closed marks (circles or triangles) represent steroid-treatedand steroid-untreated patients, respectively. MS = multiple sclerosis; CNS-infl = patients with inflammatory diseases in the central nervous system other than MS.
Leu8+ suppressor-inducer subset in the CNS. Accumulating evidence has suggested that CD4 + lymphocytes lose highly expressed CD45R antigen when activated by stimulation with phytohemagglutinin 11517}, whereas the Leu8 antigen does not disappear permanently after prior activation {l8}. During this process, the CD4+ lymphocytes gain high densities of CDw29 1151 and UCHLl {l6} antigens. Since this phenotypic change was also observed in the CD8+ cell population { 151, the predominance of CD45R- cells in the CSF may indicate a sequestration of onceactivated lymphocytes into the CNS in a noninflammatory condition. This assumption agrees with the report that more than 80% of lymphocytes were both CDw29+ and UCHLl in CSF obtained from healthy volunteers 1191. A concordant finding is that numerous T a l + cells reside in the CSF of patients with NINDs 1203; the T a l antigen is a marker for previously activated T cells 121). It has been suggested that lymphocytes enter the CNS through postcapillary venules {I}, and only activated T cells can invade vas+
Matsui et al: Immunoregulation in the CNS 649
cular endothelium by degrading subendothelial extracellular matrix 122, 23). Thus, it is likely that activated T cells selectively cross the BBB and circulate through the CNS. An alternative interpretation, however, is that the CD45R-CDw29+UCHLl+ subset has a greater ability than CD45R+ cells to adhere to endothelial cells 1241. It is noteworthy that CD4+ CD45R-Leu8 lymphocytes are potent producers of interleukin-2 (IL-2) and interferon gamma upon activation 125, 261. Therefore, a relative enrichment of this type of cell in the CSF, although small in absolute number, seems to promote a swift immune response to target antigens. In the present study, the proportion of the CD4+ CD45R- cells in the CSF was significantly elevated in the active stage of relapsing-remitting MS, irrespective of ongoing steroid treatments. Although we classified MS disease activity according to time from neurological relapse without examining for new MS plaques by magnetic resonance imaging 1271, our criteria sufficed to select patients with active MS. The increased CD4+CD45R- cells appear to consist mainly of cells actively entering the CNS rather than those proliferating within the CNS, since IL-2 receptor-positive T cells have rarely been seen in the CSF or in the inflammatory lesions of MS 120, 28). By contrast, CD4CD45R+ cells were increased in the CSF of the patients with CNS-infl. A similar finding was that 2H4+ cells were numerous in tissues of viral encephalitis C291. Thus, there may be different patterns of cellular immune reaction in the CNS, according to the types of CNS inflammatory conditions. Further clarification of the specific functions of the subsets of lymphocytes compartmentalized within the CNS could serve as an important clue to immunoregulatory therapy for inflammatory CNS diseases.
This work was supported in part by a grant from the Ministry of Health and Welfare of Japan, and by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan (No. 01770542). We thank Mr S. Araya for technical assistance.
References 1. Leibowitz S, Hughes RAC. Immunity and the blood-brain barrier. In: Turk J, ed. Immunology of the nervous system. Current topics in immunology, vol 17. London: Arnold, 1983:l-19 2. Kam-Hanson S, Link H, Fryden A, Moller E. Reduced in vitro response of CSF lymphocytes to mitogen stimulation in multiple sclerosis. Scand J Immunol 1979;10:161-169 3. Hafler DA, Weiner HL. T cells in multiple sclerosis and inflammatory central nervous system diseases. Immunol Rev 1987; 100:307-332 4. Wekerle H , Lmington C, Lassmann H , Meyermann R. Cellular immune reactivity within the CNS. Trends Neurosci 1986;9: 271-277
650 Annals of Neurology Vol 27 No 6 June 1990
5. Morimoto C, Letvin NL, Distaso J, et al. The isolation and characterization of the human suppressor inducer T cell subset. J Immunol 1985;134;1508-15 15 6. Damle NK, Mohagheghpour N, Engleman EG. Soluble antigen-primed inducer T cells activate antigen-specific suppressor T cells in the absence of antigen-pulsed accessory cells: phenotypic definition of suppressor-inducer and suppressor-effector cells. J Immunol 1984;132:644-650 7. Kansas GS, Wood GS, Fishwild DM, Engleman EG. Functional characterization of human T lymphocyte subsets distinguished by monoclonal anti-Leu-8. J Immunol 1985;134:2995-3002 8. Landay A, Gartland GL, Clement LT. Characterization of a phenotypically distinct subpopulation of Leu-2 + cells that suppresses T cell proliferative responses. J Immunol 1983;131: 2757-2761 9. Clement LT, Dagg MK, Landay A. Characterization of human lymphocyte subpopulations: alloreactive cytoroxic T-lymphocyte precursor and effector cells are phenotypically distinct from Leu2+ suppressor cells. J Clin Immunol 1984;4:395-402 10. Poser CM, Paty DW, Scheinberg L, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983;13:227-23 1 11. Matsui M, Mori KJ, Saida T, et al. The imbalance in CSF T cell subsets in active multiple sclerosis. Acta Neurol Scand 1988; 77:202-209 12. Lanier LL. Non-lineage, LFA-1 family, and leukocyte common antigens. In: McMichael AJ, Beverley PCL, Cobbold S, et al, eds. Leukocyte typing 111. White cell differentiation antigens. Oxford: Oxford University Press, 1987:796-800 13. Morimoto C, Letvin NL, Boyd AW, et al. The isolation and characterization of the human helper inducer T cell subset. J Immunol 1985;134:3762-3769 14. Takeuchi T, DiMaggio M, Levine H, et al. C D l l molecule defines two types of suppressor cells within the T8+ population. Cell Immunol 1988;111:398-409 15. Serra HM, Krowka JF, Ledbetter JA, Pilarski LM. Loss of CD45R (Lp220) represents a post-thymic T cell differentiation event. J Immunol 1988;140:1435-1441 16. Akbar AN, Terry L, Timms A, et al. Loss of CD45R and gain of UCHLl reactivity is a feature of primed T cells. J Immunol 1988;140:2171-2178 17. Clement LT, Yamashita N, Martin AM. The functionally distinct subpopulations of CD4 + helperhnducer T lymphocytes defined by anti-CD45R antibodies derive sequentially from a differentiation pathway that is regulated by activation-dependent post-thymic differentiation. J Immunol 1988;141:14641470 18. Kanof ME, James SP. Leu-8 antigen expression is dininished during cell activation but does not correlate with effector function of activated T lymphocytes. J Immunol 1988;140:37013706 19. Hedlund G, Sandberg-Wollheim M, Sjogren HO. Increased proportion of CD4+CDw29+CD45R-UCHL-l+ lymphocytes in the cerebrospinal fluid of both multiple sclerosis patients and healthy individuals. Cell Immunol 1989;118:406-4 12 20. Hafler DA, Fox DA, Manning ME, et al. In vitro activated T lymphocytes in the peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. N Engl J Med 1985;312:14051411 21. Hafler DA, Fox DA, Benjamin D, Weiner HL. Antigen reactive memory T cells are defined by Tal. J Immunol 1986;137: 4 14-4 18 22. Naparstek Y, Cohen IR, Fuks Z , Vlodavsky I. Activated T lymphocytes produce a matrix-degrading heparan sulphate endoglycosidase. Nature 1984;310:241-244 23. Savion N, Vlodavsky I, Fuks 2. Interaction of T lymphocytes
and macrophages with cultured vascular endothelial cells: attachment, invasion, and subsequent degradation of the subendothelid extracellular matrix. J Cell Physiol 1984;118:169-178 24. Pittalis C, Kingsley G, Haskard D, Panayi G. The preferential accumulation of helper-inducer T lymphocytes in inflammatory lesions: evidence for regulation by selective endothelial and homotypic adhesion. Eur J Immunol 1988;18:1397-1404 25. Dohlsten M, Hedlund G, Sjogren H - 0 , Carlsson R. Two subsets of human CD4+ T helper cells differing in kinetics and capacities to produce interleukin 2 and interferon-? can be defined by the Leu-18 and UCHLl monoclonal antibodies. Eur J Immunol 1988;18:1173- 1178 26. Hedlund G, Dohlsten M, Sjogren HO, Carlsson R. Maximal
interferon-gamma production and early synthesis of interleukin2 by CD4+CDw29-CD45R-p80- humanT lymphocytes. Immunology 1989;66:49-53 27. Isaac C, Li DKB, Genton M, et al. Multiple sclerosis: a serial study using MRI in relapsing patients. Neurology 1988;38: 1511-1515 28. Hayashi T,Morimoto C, Burks JS, et al. Dual-label immunocytochemistry of the active multiple sclerosis lesion: major histocompatibility complex and activation antigens. Ann Neurol 1988;24:523-531 29. Sobel RA, Hafler DA, Castro EE, et al. The 2H4 (CD45R) antigen is selectively decreased in multiple sclerosis lesions. J Immunol 1988;140:22 10-2214
Matsui et al: Immunoregulation in the CNS
651
